Particles

ABSTRACT

A coated magnetic particle comprising an optionally porous magnetic polymer particle of a matrix polymer, said polymer particle having on a surface and/or in the pores thereof superparamagnetic crystals, said coated particle having a coat formed of a coating polymer, wherein said coated magnetic particle is essentially non-autofluorescent.

This invention relates to magnetic polymer particles, to processes fortheir preparation and to their uses. In particular, the inventionrelates to coated magnetic polymer particles which exhibit lowautofluorescence.

Magnetic polymer particles are of general utility in various medical andbiochemical fields, for example as transport vehicles for the deliveryof pharmaceutical products, for diagnostic purposes, for separation andfor synthetic purposes. Such particles rely upon their magneticproperties in order to perform these functions: in diagnostic assayapplications, for example, application of a magnetic field to a samplecontaining an analyte bound to magnetic polymer particles allows theisolation of the analyte without the use of centrifugation orfiltration; and in therapeutic applications, for example, application ofa magnetic field to the patient may serve to target drug-carryingmagnetic polymer particles to a desired body site.

By magnetic is meant herein that the polymer particles containsuperparamagnetic crystals. Thus the magnetic polymer particles' aremagnetically displaceable but are not permanently magnetizable.

Many processes for preparing magnetic polymer particles are known, alarge number of which involve preparing maghemite- ormagnetite-containing polymer particles from pre-formed magnetic ironoxides, e.g. magnetite. Some of processes involved are described in U.S.Pat. No. 4,654,267 (Ugelstad) the contents of which are incorporatedherein by reference.

Thus U.S. Pat. No. 4,654,267 outlines a number of limitations withregard to the processes which preceded it; these include difficulty inobtaining magnetic particles of similar size and/or of homogeneous oruniform magnetic properties, as well as a more general problem relatingto the difficulty of incorporating magnetic material inside the cavitiesof porous polymer particles.

With deposition taking place principally on the surface, or in largeopen cavities, leaching of magnetic particles, which shortens the usefullifetime of magnetic polymer particles in the applications to which theyare put, was consequently problematic.

In order to overcome these disadvantages, to produce porous magneticpolymer particles having magnetic material disposed within the polymerpores, U.S. Pat. No. 4,654,267 advocated the use of porous polymerparticles having surface functional groups which serve to draw the ironions into the polymer particles. These functional groups could eitherresult from the use of functionalized comonomers in the production ofthe polymer or from post-polymerization treatment of the polymer tointroduce the functional groups, e.g. by coupling to or transformationof existing groups on the polymer surface.

The leaching of superparamagnetic crystals from the porous polymerparticles may be further inhibited by forming a further polymer coatingover the superparamagnetic crystal loaded polymer particles, or moreparticularly by at least partly filling the pores of the particles witha polymer coating. Such coating polymers may typically be formed frommonomers reactive with functional groups pendant from the surface of thepolymer of the underlying particles.

However, when coated magnetic polymer particles are prepared in this waythey exhibit the characteristic known as autofluorescence, i.e. theyexhibit intrinsic fluorescence. While for many of the uses of coatedmagnetic polymer particles, e.g. where they are used in syntheses orseparations, autofluorescence does not cause problems, where theparticles are to be used in assays in which light emissions aredetected, autofluorescence by the particles can produce unwantedbackground signals. The autofluorsecence may also mask the signal froman emitter of interest. This will decrease the sensitivity of the assay.

We have now surprisingly found that the autofluorescence exhibited by amagnetic polymer particle can be minimised by the avoidance ofconjugated delocalised electron systems (other than those found inbenzene rings) in the matrix polymer, in the particle coating and in anysurface functionalities. Such conjugated delocalised electron systemshad previously been considered essential to allow attraction of ironoxides in the magnetisation step and polymer coating to occursuccessfully.

As a result, a matrix polymer with reduced or no tendency toautofluorescence can be used in the preparation of coated magneticpolymer particles which therefore exhibit remarkably lowautofluorescence. Such particles may have utility in a variety ofassays, in particular assays in which fluorescence is detected.

Whilst the use of fluorescent entities as labels in non-magnetic polymerbeads is known, the use of such fluorescent labels in magnetic beads hasthe advantage of enabling the combination of magnetic sample preparationand detection, i.e. combining instrumentation for automated samplepreparation and/or concentration with detection platforms such as flowcytometers, fluorescence microscopes, high resolution array scanners orlaser scanning cytometers. These detection devices can all be includedin microfluidic devices where the possibility of using the magneticproperties for sample preparation and/or concentration will beimportant.

Thus viewed from one aspect the invention provides an optionally porousmagnetic polymer particle comprising a matrix polymer, with disposed onthe surface and/or in the pores thereof superparamagnetic crystals, saidmatrix polymer preferably being essentially free of conjugateddelocalized electron systems and optionally being surface functionalisedwith groups selected from sulphonic acid, carboxylic acid, amine, andepoxy groups, said particle being essentially non-autofluorescent, e.g.having a level of autofluorescence corresponding to a difference in theMean Grey Value (ΔMGV) at an excitation wavelength of 450±25 nm for anexposure time of 500 ms or 600 ms of less than 600.

Viewed from another aspect the invention provides a coated magneticparticle comprising an optionally porous magnetic polymer particle of amatrix polymer, said polymer particle having on a surface and/or in thepores thereof superparamagnetic crystals, said coated particle having acoat formed of a coating polymer, wherein said coated magnetic particleis essentially non-autofluorescent, e.g. has a level of autofluorescencecorresponding to a difference in the Mean Grey Value (ΔMGV) at anexcitation wavelength of 450±25 nm for an exposure time of 500 ms or 600ms of less than 600 and said coated particle is preferably essentiallyfree of conjugated delocalized electron systems.

By matrix polymer (or polymers) is meant the polymer (or polymers) and,if required, cross-linking agents employed to form the uncoated polymerparticle, as further defined in detail below.

By essentially free of conjugated delocalized electron systems is meantthat the matrix polymer and if present said coating polymer (i.e. thecoated particle itself) do not contain any electronic systems in whichdelocalisation occurs (other than within a benzene ring).

The uncoated magnetic polymer particles produced by formingsuperparamagnetic crystals in the pores of porous matrix polymers,optionally surface functionalized e.g. with sulphonic acid, carboxylicacid, amine, or epoxy groups (but not with nitro groups), should beeffectively non-autofluorescent. Such uncoated particles (and hence thecoated particles) preferably have ΔMGV values of less than 300,especially less than 200, particularly less than 100. Such uncoatedparticles may be used as intermediates in the preparation of the coatedparticles of the invention or in end uses where leaching of thesuperparamagnetic crystals is not problematic.

The ΔMGV for the uncoated and coated particles is more preferably lessthan 400, particularly below 350, more particularly below 300.

The ΔMGV for the particles is determined using a fluorescence microscopeby illumination of a substrate (e.g. glass) surface carrying theparticles with light from a 100 W mercury or 75 W xenon lamp filteredthrough a 450±25 nm excitation band pass filter, with a beam splitter at480 nm and with the emitted light being filtered through a 520 nm longpass filter. ΔMGV is the difference in the Mean Grey Value determinedfor a minimum of 30 particles and the Mean Grey Value determined for asimilar minimum of background positions (i.e. positions on thesubstrate) e.g. 25 positions.

Conveniently ΔMGV can be determined using a BX-61 fluorescencemicroscope from Olympus equipped with a 100 W mercury or 75 W xenon lampand excitation and emission filters from Chroma Technology and anUplan-Apo 20× objective. Emission detection can be effected using anF-view digital monochrome CCD camera (4096 grey levels) for an exposuretime of 500 ms (Xenon lamp) or 600 ms (mercury lamp) and image analysisto determine the MGV values may be performed using the Analysis softwareof Soft Imaging Systems.

For autofluorescence determination the particles are either dispersedbetween parallel glass substrates (e.g. microscope slides) in a liquidmedium, e.g. 1:1 by volume mixture of glycerol and 1.5 g/l sodiumdodecyl sulphate in water (as in Example 12) or buffered in pH 7.4phosphate buffer saline (Example 18). Autofluorescence measurement isexplained in detail in examples 12 and 18.

The superparamagnetic crystals in the polymer particles used in theparticles of the invention may be of any material capable of beingdeposited in superparamagnetic crystalline form on the surface of apolymer particle or more preferably in the pores of a porous polymerparticle. Thus the superparamagnetic crystals in the particles of theinvention may be of any element, alloy or compound capable of exhibitingsuperparamagnetism. Magnetic iron oxides, e.g. magnetite or maghemiteare preferred; however the crystals may be of mixed metal oxides orother magnetic material if desired. The total quantity of crystallinemagnetic material present is generally more than 1%, preferably morethan 3%, desirably more than or equal to 5% (by weight, e.g. up to 40%wt. The percentage is calculated on a Fe (or equivalent metal in thecase of magnetic materials other than iron oxides) weight basis basedupon the overall dry weight of the coated particles. Thesuperparamagnetic crystals will be on a surface of the matrix polymer,i.e. on the external surface of the particle or, more preferably, on asurface of a pore in a porous matrix particle.

The optionally porous magnetic polymer particle may be prepared byconventional means using monomers. In general any porousnon-autofluorescent polymer particles can be used. However, the monomersused in forming the matrix polymer (and the coating polymer) shouldpreferably be such that the resultant polymer is substantially free fromconjugated delocalized electron systems other than benzene rings. Thusfor example the use of divinylbenzene as a cross-linking agent shouldpreferably be avoided.

Also the presence of nitro groups on the surface of the polymer particleshould be avoided since such groups inherently contain delocalisedelectron systems and therefore contribute to the autofluorescence of thepolymer particle.

The matrix polymer is especially preferably vinylic, a styrenic or,especially, an acrylic polymer. Preferred monomers for matrix polymerpreparation include methyl methacrylate, methacrylic acid, hydroxyethylmethacrylate, glycidyl methacrylate, butylmethacrylate, acrylic acid,ethyleneglycol dimethacrylate, trimethylolpropane trimethacrylate,trimethylol triacrylate, pentaerythritol tetraacrylate and other acrylicor methacrylic monomers.

The matrix polymer is preferably not water-swellable.

In one preferred embodiment of the invention, the matrix polymer may beproduced by a seeded polymerization reaction, i.e. a multistagepolymerization in which the particulate polymer from one stage is usedas a seed for a subsequent polymerization reaction in which a largerdimension particulate is produced. In this embodiment, the monomers usedin one stage need not be the same as those used in the other stage(s).In this embodiment, the polymer produced in the final stage ispreferably an acrylic polymer. The polymer produced in the initialpolymerization stage is preferably a styrenic polymer. In an especiallypreferred embodiment the initial polymer (the seed) is styrenic, anintermediate polymer is a styrenic/acrylic copolymer, and the finalpolymer is acrylic.

The matrix polymer containing particles may be prepared by any techniquesuitable for producing particulate polymer particles, e.g. suspensionpolymerization, emulsion polymerization, dispersion polymerisation etc.However, the particles are especially preferably produced using the wellknown Ugelstad process, e.g. as described in U.S. Pat. No. 4,654,267 orWO00/61647. Preferably however, ammonia is employed in the magnetisationprocess instead of ethylene diamine. The use of ammonia has beensurprisingly found to give rise to particles having lower levels ofautofluorescence.

Thus, prior to incorporation of the magnetic particles it is possible tointroduce amino groups onto the particle surface using ammonia.

Particles may be produced using the Ugelstad process which aresubstantially monodisperse, i.e. the coefficient of variation (CV) ofthe particle diameter is very small, e.g. 2 to 5%. CV is defined as 100times (standard deviation) divided by mean where mean is mean particlediameter and standard deviation is standard deviation in particle size.CV is preferably calculated on the main mode, ie. by fitting a monomodaldistribution curve to the detected particle size distribution. Thus someparticles below or above mode size may be discounted in the calculationwhich may for example be based on about 90% of total particle number (ofdetectable particles that is). Such a determination of CV is performableon a Coulter LS 130 particle size analyzer.

The coating polymer is preferably formed from at least one epoxidecompound, preferably at least two epoxide compounds.

The reaction of the porous magnetic polymer particle with the coatingmonomers generates a coating polymer within the pores of the matrixpolymer particles which serves essentially to block these pores,physically encapsulating the superparamagnetic crystals within thepolymer particles. The resulting “coated” particles then have reducedporosity relative to the porous starting material. Surprisingly it isbelieved that the superparamagnetic crystals appear to catalyse thepolymerization so that the coating forms preferentially in theirvicinity. Since the majority of the superparamagnetic crystals arewithin pores in the starting porous particles, the coating may not formto any significant extent on the external surface of the particles.

In one preferred embodiment, the porous polymer particles are reactedwith a mixture of epoxides. Preferably, the epoxides contain at leastone ether link and optionally a hydrophobic component, e.g. a alkylenechain. Generally the epoxides will have a carbon atom content of from 3to 50, preferably 3 to 25. Typical epoxides that may be used includeepichlorohydrin, epibromohydrin, isopropylglycidyl ether, butyl glycidylether, allylglycidyl ether, 1,4-butanediol diglycidylether(1,4-bis(2,3-epoxypropoxy)butane), neopentylglycol diglycidylether, ethylene glycol diglycidyl ether, glycerol diglycidyl ether,glycidol, and glycidyl methacrylate, ethyl hexyl glycidylether, methylglycidylether, glycerol propoxylate triglycidylether, poly(propyleneglycol) diclycidylether, 1,3 butanediol diglycidylether, tert-butylglycidylether, 1,4 cyclohexanedimethanol diglycidyl ether, diethyleneglycol diglycidyl ether, dodecyl glycidylether, 0-(2,3epoxypropyl)-O′-methylpolyethylene glycol glydidylether, glycidyltetrafluoroethyl ether, 1,6 hexanediol diglycidylether, octylglycidylether, decyl glycidylether, poly(epichlorohydrin-co-ethyleneoxide-co-allyl glycidylether), polyethylene glycol diglycidyl ether,trimethylolethane triglycidylether, trimethylolpropan, triglycidylether,tert-butyldimehytlsilyl glycidylether, 1,2-epoxybutane,1,2-epoxypentane, 1,2-epoxy-5-hexene, 1,2-epoxy-hexane,1,2-epoxy-7-octene, 1,2-epoxyoctane, 1,2,7,8-Diepoxyoctane,1,2-Epoxy-9-decene, 1,2-Epoxydecane, 1,2-Epoxydodecane,1,2-Epoxytetradecane etc.

Typically, the coating reaction may be effected by impregnating theporous magnetic polymer particle with the coating monomers e.g. using asolution of these (for example in an organic solvent such as methanol,toluene, xylene, diethylenglycol, dimethyl ether or diglyme) or bymixing a dispersion of the porous particles in an organic solvent with aliquid epoxide mixture. Sonication may be used to improve impregnationand the reaction may be accelerated by raising the temperature, e.g. to50-100° C. Any solvent used may be extracted by application ofsub-ambient pressure.

Viewed from a further aspect the invention provides a process for thepreparation of coated magnetic particles as herein before describedbeing free of conjugated delocalised electron systems, said methodcomprising reacting porous magnetic polymer particles essentially freefrom surface nitro groups and having superparamagnetic crystals on thesurface and/or in the pores thereof with polymer-forming monomersselected from epoxides.

Viewed from a further aspect the invention provides the use of suchparticles in syntheses, extractions or assays.

If desired further materials may be impregnated into or chemicallyattached to the particles either before the coating polymerizationreaction or after coating polymerization. Typically such furthermaterials will be radiation emitters or absorbers, e.g. fluorophores,material giving luminescence, material giving time delayed fluorescence,chromophores or radioactively labelled materials.

Particles according to the present invention will generally have sizes(i.e. diameters) that are generally in the micrometer range, e.g. 0.3 to100 μm, especially 0.5 to 50 μm, more especially 0.8 to 10 μm, e.g. 2 to10 μm, e.g. 5 and 8 μm.

Typically the porous particles used will have a surface area of at least15 m²/g (measured by the BET nitrogen absorption method), and morepreferably at least 30 m²/g, e.g. up to 700 m²/g, when corrected to amean particle diameter of 2.7 μm (i.e. multiply surface area by 2.7/MD,where MD is the mean diameter in micrometers). Similarly scaled, theparticle pore volume is preferably at least 0.1 mL/g.

Typically, the polymer particles are spherical and substantiallymonodisperse before they are coated and especially preferably remainspherical and substantially monodisperse once they have been coated.

By substantially monodisperse it is meant that for a plurality ofparticles (e.g. at least 100, more preferably at least 1000) theparticles have a coefficient of variation (CV) of less than 20%, forexample less than 15%, preferably less than 12%, more preferably lessthan 11%, still more preferably less than 10% and most preferably nomore than about 8%, e.g. 2 to 5%.

Generally, the uses to which magnetic polymer particles are put, e.g.their use as diagnostic tools, require an appropriate degree ofelectrophilicity in order that they may participate adequately incoupling and other reactions in aqueous systems prevalent in biologicalmedia.

Whilst the general polarity of the coatings is desirably electrophilic,certain coatings which contain hydrophobic moieties may be incorporatedso as to tailor the degree of electrophilicity to that which is desired.In this way, the invention permits the provision of useful diagnosticand other tools having a wide range of polarities.

If desired the surfaces of the coated magnetic polymer particles may befurther functionalised, e.g. by coupling a drug molecule, a reporterlabel (e.g. a chromophore, enzyme or radiolabel), or an affinity ligand(e.g. an antibody or antibody fragment, a member of a specific bindingpartner pair (e.g. biotin or streptavidin), an oligopeptide, anoligonucleotide, or an oligosaccharide).

Such coupling may be direct or indirect (and so may or may not involvethe use of a coupling agent to form a linkage between the particle andthe substance being coupled to it) and may be biodegradable ornon-biodegradable. Biodegradable couplings may be desired if themagnetic polymer particles are to be used for the targeted release of anactive compound. Accordingly after coating has been effected, thependant groups of the coating may be manipulated to provide appropriatefunctionality (for example epoxy, hydroxy, amino, ketones, mercapto,isothiocyanate, isocyanate, tosyl, carboxylic acid etc functionalities)for the attachment of such substances.

The low autofluorescence particles of the invention are of particularuse in applications where the fluorescence of the particle is to bedetected, e.g. in an assay where a reporter carries a fluorophore. Byfluorophore is meant a compound which exhibits intrinsic fluorescence.In some assays it may be preferable to employ coloured particles orbeads. In this regard, coloured particles/beads for use in such assaysrequire the presence of a fluorophore to “colour code” the particle.Many different fluorophores are known allowing the preparation of avariety of colour coded beads, (i.e. each bead class emitting light atdistinct wavelengths, different intensity etc when excited by light of alower wavelength. The excitation source can be laser(s) or filteredlight of one or several wavelengths). Such coding may be constructed by,for example, providing beads of differing fluorescence intensity, withdiffering fluorescence spectra, differing intensity-ratio between two ormore fluorophores, or by using beads of differing diameters with variousfluorophores. Other possible coding methods may be use of magnetic tags,refractive index, adsorption spectrums, adsorption ratios etc.

Fluorophores can be attached to the low autofluorescence particles ofthe invention by, for example, contacting a particle having surfacefunctional groups, e.g. amino groups, with a fluorophore comprising areactive group forming covalent attachment of the fluorophore to theparticles. Such functional groups may be succinimidyl esters,isothiocyanates, carboxylic acids, maleimides, sulphonyl chlorides etc.

Other potential methods for incorporation of fluorophores onto (or into)the beads may involve hydrophobic or electrostatic interactions,swelling of the fluorophores into the coating and entrapment (orcapture) of the fluorophores in the coating (entrapment may be inducedby change of polarity of the solvents used), deposition of fluorophoresin pores, swelling fluorophores into polymer matrix etc.

The attachment of the fluorophore to the particle can be achieved at anysuitable stage of the particle synthesis. Thus, a fluorophore may beattached to the particle after the magnetisation process but before acoat is subsequently applied. Alternatively, a thin coat may be appliedto the particle prior to the fluorophore being added and a subsequentfurther coat applied. Most conveniently however, the fluorophore isattached to the particle after the final coat has been applied.

The latter process is most favoured since the application of a coat overthe fluorophore may significantly reduce its fluorescent intensityalthough for the coumarin dye AMCA (from Molecular Probes Inc.)intensity reduction is not observed if a coating is placed over it.

The reaction between the particle and the fluorophore shouldconveniently take place in an organic solvent such as dimethylformamide,acetonitrile, acetone, 1,4-dioxane, chloroform, methanol, ethanol,toluene, butylacetate, ethylacetate etc alone or mixtures thereof; whatis primarily required is a solvent system which allows a homogenousdistribution of dye to all the particles and allows incorporation of dyein high yield. This will be readily carried out by the person skilled inthe art.

The fluorophore functionalised coated magnetic particle should alsocarry an affinity ligand, the nature of which will be selected based onits affinity for a particular analyte whose presence or absence in asample is to be ascertained. The affinity ligand may therefore compriseany molecule capable of being linked to a magnetic probe which is alsocapable of specific recognition of a particular analyte. Affinityligands therefore include monoclonal antibodies, polyclonal antibodies,antibody fragments, nucleic acids, oligonucleotides, proteins,oligopeptides, polysaccharides, sugars, peptides, peptide encodingnucleic acid molecules, antigens, drugs and other ligands. Most commonlythe affinity ligand is an antibody which binds to an antigen in asample. Examples of suitable affinity ligands are available in thepublished literature and are well known. The use of further bindingpartners, secondary affinity ligands and linking groups is routine inthe art and it will be appreciated that the use of such species with theparticles of the invention is possible if desired.

More specifically, nucleic acid detection generally involves probing asample thought to contain target nucleic acids using a nucleic acidprobe that contains a nucleic acid sequence that specificallyrecognises, e.g. hybridises with, the sequence of the target nucleicacids, such that the nucleic acid affinity ligand and the target nucleicacids in combination create a hybridisation layer. Correspondingly,immunoassay type detection of hormones, antibodies, cytokines etcgenerally involves binding of an antibody or an antigen to the beads. Itmay also be of interest to bind ligands that have specific affinity for“tags” which have been introduced on the target of interest. Suchligands can be a metal chelate, small organic molecule, nucleic acidderivative or protein. For applications in cellular biology andmicrobiology it will be of interest to bind antibodies with affinity forspecial cell types, bacteria or viruses.

The target material is optionally a biological molecule or molecule ofsynthetic origin, or conjugation of the two types, e.g. it may be amolecule or a group of molecules, cells or a single cell organism or avirus including for example antibodies, amino acids, proteins, peptides,polypeptides, enzymes, enzyme substrates, hormones, lymphokines,metabolites, antigens, haptens, cytokines, lectins, avidin,streptavidin, toxins, poisons, environmental pollutants, carbohydrates,oligosaccharides, polysaccharides, glycoproteins, glycolipids,nucleotides, oligonucleotides, nucleic acids and derivatised nucleicacids, DNA, RNA, natural or synthetic drugs, receptors, virus particles,bacterial particles virus components, cells, cellular components,natural or synthetic lipid vesicles, polymer membranes, polymer servicesand particles and glass and plastic surfaces.

As well as being coupled to such target-binding ligands, the particlesproduced according to the invention may also be subsequently labelledwith a reporter moiety, e.g. biotin, a chromophore, fluorophore,radioisotope or enzyme.

Of particular further interest is the possibility of including afluorophore as a reporter moiety especially in conjunction with a colourcoded particle as described above. The incorporation of a fluorophoreinto a reporter molecule is known in the art.

For example, in nucleic acid detection, an oligonucleotide probecomplementary to the target, labelled with one or several fluorophorescan be employed. Labelling may be achieved by e.g. using modifiedphosphoramidites during probe synthesis.

Alternatively, enzymatic incorporation of labelled nucleotdies or probescan be employed when the target is present.

For protein/immunoassays, an antibody or antigen can be labelled usingstandard cross-linking reagents; e.g. N-hydroxybuccinimide activatedfluorophores which react with amino groups on the protein or antigen.

When such a reporter is combined with a low autofluorescence particle ofthe invention already attached to a “colour coding” fluorophore thepresence of two excitable fluorophores exists. This allows the beads tobe utilised in a variety of assays as described in more detail below andallows multiplexing.

The fluorophores utilised should be chosen such that their excitationand emission signals do not interfere. Whilst the person skilled in theart can tailor the fluorophores they use to minimise overlap, becausemany fluorophores have a absorption spectrum which exhibit a tail atwavelengths shorter than the absorption maximum and further to avoidexcitation of the code with the excitation light intended for thereporter, preferably the code (i.e. particle fluorophore) should be setat shorter wavelengths than the reporter. Moreover, the shorterwavelength region (blue and green emission) is also the region of thespectrum where inherent autofluorescence is most significant.

In a particularly preferred embodiment of the invention therefore, thefluorophore attached to the particle will emit in the blue region of thespectrum (i.e. at shorter wavelengths that the reporter) whereas thefluorophore attached to the reporter moiety will emit in the red regionof the spectrum (i.e. at longer wavelengths than the particlefluorophore).

Alternatively viewed, the colour fluorophore (code) attached to theparticle will emit at shorter wavelengths than the reporter whereas thefluorophore attached to the reporter moiety will emit at longerwavelengths than the code.

Suitable fluorophores of use are listed below:7-Hydroxy-4-methylcoumarin (AMCA/AMCA-X), Alexa fluor 350, Alexa fluor405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546,Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647,BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY576/589, BODIPY 581/591, BODIPY 630/650-X, BODIPY 650/665-X, BODIPY FL,BODIPY TMR, BODIPY TR, Cascade Blue, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7,DY-505, DY-555, DY-647, Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, Pyrene, Tetramethylrhodamine, Fluorescein, with orwithout suitable functional groups and with or without a linker betweenthe fluorophore and the functional group. The fluorophores may haveappropriate functional groups e.g. amino, succinimidylester,isothiocyanates, maleimide, sulphonyl chloride, carboxylic acid etc.

Such fluorescently labelled particles can be used in various assays e.g.multiplexed immunoassays. For example, a mixture of particles withdifferent colour codings and corresponding affinity ligands may be addedto a sample to be analysed. The analyte of interest will bind byaffinity to its corresponding ligand on the coloured magnetic particles.

In a sandwich type immunoassay, a label (e.g. a fluorophore) bound to asecond antibody (affinity ligand) specific to the analyte is then boundto the captured analyte. The label may be selected from the group ofwell-known labels such as radioimmunoassay, fluorescent orchemiluminescence immunoassay, or immunoPCR technology. Extensivediscussion of the known immunoassay techniques is not required heresince these are known to those of skilled in the art.” Jackowski,George; et al., US Patent application 20020160425, Oct. 31, 2002).

The magnetic properties of the particle are used to facilitate simple(automated) washing of the bound complex in order to remove any unboundlabel and other reagents that will interfere during the detection step.The degree of interference in the absence of a washing step will dependon the nature of the system used but in most cases it is essential toremove any unbound label. In all cases, the result of washing is loweredbackground and increased signal to noise ratio compared to an analysiswithout washing.

The particle-analyte-label complex is then subjected to analysis in oneor two detection systems where a) the colour of the beads is detectedand b) the label bound is detected. This can be done simultaneously inthe same detector, in two parallel detectors or successively by usingthe same detector twice or by moving the bead complex from one detectionsystem to another. In the latter case it must be possible to trackparticles so that there is a direct link between the particle colour andlabel detection.

Multiplexing is achieved by the colour coding of the particles. Byidentifying which particle type has a signal from an associated label,the analyte can be identified. Using two or more different labels fordetection of analytes can increase the level of multiplexing.

The detection system must be able to analyse single particles. This canbe done in a flow based system such as a microfluidic flow cytometer orin an immobilised bead array format. In the later case, the beads aredeposited passively or actively by affinity or magnetic force to a solidsurface such as a microscope slide, bottom of microtitre well orstructure in a microfluidic system. The colour coding of the beads canbe read using a high resolution scanner while detection of labels on thesurface of the beads will need more sophisticated equipment such as amicroscope based image analysis system.

The detection limit of the system is determined by the detector'sability to identify labels on the surface of the beads. The sensitivitycan thus be increased if more labels are introduced per analyte or thelabel introduced is very large (in the sense easy to detect).

In competitive assays, an added labelled (e.g. with a fluorophore)analyte competing with the analyte of interest is employed. An affinityligand (antibody) with equal affinity for the natural analyte and thelabelled analyte is immobilized on the colour coded beads, so that eachset of colour coded beads has specific affinity for one set of analytes.

The assays contemplated can utilise bead array technology. In a beadarray format, particles can be (passively) spotted or positioned inconfined areas on a surface using magnetism. The results of the beadarray are read in a two-step method in which the beads are decoded. Theposition of each bead and its colour coding is recorded by a suitablesoftware and the signal from positive beads is recorded and correlatedwith the nature of its coding.

Alternatively, a labelled reporter need not be used and instead theanalyte may be further reacted in such a way that it is bound by biotinor a/biotinylated molecule is bound to the bead/analyte complex. Thiscan then be bound to a streptavidin coated slide and analysed bymicroscopy or laser scanning.

In a microfluidic competitive multiplexed immunoassay the bead may actas both reporter and label. Beads may be coated with an antibody againstthe target antigen. Multiplexing is achieved by using one colour codingfor each antibody-antigen pair of interest. A standard competitive assaymay then be carried out in the presence of a biotinylated antigen. Thiscan be achieved in any suitable vessel, e.g. a tube, well, or amicrofluidic device.

After incubation, magnetic separation is used to wash the bead-antigencomplex free of unincorporated antigens. The samples are then passedover a streptavidin coated surface. Using optimised flow or washingconditions, only the bead complexes with competitive, biotinylatedantigens are bound. Thus, samples containing high concentration of atarget antigen will have low or no beads with the corresponding colourcoding bound, while there will be a high number of beads bound of acolour coding corresponding to a low concentration target.

The microfluidic device can be a simple as a defined area on amicroscope slide, and can be made by microarray spotting ofstreptavidin. Alternatively, more complex solutions can be madeincorporating mixing, magnetic separation and detection in themicrofluidic device.

Thus, viewed from another aspect the invention provides use of theparticles of the invention in an assay which comprises the steps of:

-   i) adding a coated magnetic particle comprising a first fluorophore    and an affinity ligand to a sample in which a target analyte may be    present;-   ii) allowing said affinity ligand to bind with said analyte if    present to form a complex;-   iii) contacting the complex with a reporter to allow binding of said    reporter to said analyte wherein said reporter optionally carries a    second fluorophore;-   iv) magnetically separating said particles from said sample; and-   v) detecting said first fluorophore and if present said second    fluorophore.    or an assay which comprises the steps of:-   i) contacting a coated magnetic particle comprising a first    fluorophore and an affinity ligand with a sample in which a target    analyte may be present and in which an analyte carrying a reporter    capable of competing with said target analyte for the affinity    ligand is present:-   ii) allowing said affinity ligand to bind to said analyte, if    present, or said analyte carrying a reporter;-   iii) magnetically separating said particles from said sample; and-   iv) detecting the first fluorophore.

Whilst assays have been described here with reference to fluorophores,it would of course be possible to use other labels in conjunction withfluorophores. For example, differently sized beads could be employed orbeads labelled with radio labels.

The invention will now be described further by reference to thefollowing Examples. These are not intended to be limitative but merelyexemplary of the invention.

In the Examples percentages are by weight unless specified otherwise.

EXAMPLE 1 Seed Latex

18.0 g of a polystyrene latex with 0.5 micrometer monosized particlesmade by emulsion polymerisation was stirred at room temperature togetherwith an emulsion of 10.8 g dioctanoyl peroxide and 10.8 g acetone in108.6 g 0.6% sodium dodecyl sulphate (SDS) in water. After 20 hours, thereaction mixture was added to an emulsion of 93.3 g methyl methacrylateand 93.3 g styrene in 859.1 g of 0.25% SDS-solution. After stirring for2 hours at 25 W, 803.4 g of a solution of 0.05% KI in water was chargedinto the reactor and the temperature was raised to 700C for 10 hours.After polymerisation, a seed latex containing 8.6% dry substance of 1.9micrometer monosized particles was obtained.

EXAMPLE 2 Porous Epoxy Particles

34.5 g of the particles of Example 1 was mixed with an emulsion of 5.9 gdioctanoyl peroxide and 5.9 g acetone in 59.1 g 0.5% SDS in water. Afterstirring for 24 hours at room temperature, the reaction mixture wasadded to an emulsion of 51.8 g glycidyl methacrylate, 51.8 g ethyleneglycol dimethacrylate and 103.6 g butyl acetate in 1013 g of a 0.1%solution of hydroxypropyl methylcellulose in water. The suspension wasstirred for 2 hours at room temperature and 474.5 g of a 0.44% solutionof hydroxypropyl methylcellulose in water was added. The temperature wasraised slowly to 70° C. and the particles were polymerised for 5 hours.After purification with methanol, monosized particles with diameters of7.5 micrometers were obtained. By nitrogen adsorption, the specificsurface area was determined to be 19.2 m²/g dry substance.Auto-fluorescent of these particles could not be observed byfluorescence microscopy or flow cytometry.

EXAMPLE 3 Superparamagnetic Particles from Epoxy Particles

3.3 g FeCl₂×4H₂O and 7.9 g FeCl₃×6H₂O were dissolved in 53 g water andadded to a dispersion of 5 g dry substance of the porous epoxy particlesof Example 2 in 61 g water. The suspension was cooled to 50C and stirredfor a few minutes before 62 ml of 25% NH₃ in water was added. Thetemperature was raised to 80° C. for 2 hours. The suspension was cooledand the particles were purified with water by several cycles ofcentrifugation. After purification, the particles were transferred tomethanol and analysed by fluorescence microscopy. No autofluorescencecould be observed under the same conditions as would show considerableautofluorescence for conventional magnetic polymer particles. The ironcontent was determined to be 67 mg Fe/g Dry substance (DS). Theparticles showed superparamagnetic behaviour and were attracted to amagnet.

EXAMPLE 4 Coating of Superparamagnetic Particles

3 g dry substance of the magnetic particles of Example 3 weretransferred to 15 g diglyme by centrifugation and resuspension. Thesuspension was stirred and 10 g butanediol diglycidyl ether and 5 gglycidol were added. The temperature was raised to 90° C. for 20 hours.The particles were purified with methanol. FT-IR absorption of 1080 cm⁻¹confirmed appearance of ether coating from the epoxides. The particleswere analysed by fluorescence microscopy. No green or redautofluorescence was observed.

The particles of Examples 6, 8 and 11 can be coated analogously.

EXAMPLE 5 Porous Particles with Sulphonic Groups

10 g dry substance of the porous epoxy particles of Example 2, suspendedin 100 ml water were mixed with 12.6 g sodium sulphite and 13.55 gsodium tetrabutyl ammonium hydrogen sulphate. The mixture was stirredfor 20 hours at 80° C. After purification with water, FT-IR absorptionof 1035 cm⁻¹ confirmed introduction of sulphonic groups.

EXAMPLE 6 Superparamagnetic Particles from Particles with SulphonicGroups

3.3 g FeCl₂×4H₂O and 7.9 g FeCl₃×6H₂O were dissolved in 53 g water andadded to a dispersion of 5 g dry substance of the particles of Example 5in 96 g water. The suspension was cooled at 5° C. and stirred for a fewminutes before 62 ml of 25% NH₃ in water was added. The temperature wasraised to 80° C. for 2 hours. The suspension was cooled and theparticles were purified with water by several cycles of centrifugation.After purification, the particles were transferred to methanol andanalysed by fluorescence microscopy. No autofluorescence could beobserved under the same conditions as would show considerableautofluorescence for conventional magnetic polymer particles. The beadswere attracted to a magnet and showed superparamagnetic behaviour. Thecontent of iron was determined to be 75.5 mg/g DS and the content ofsulphur was determined to be 0.28 mmol/g DS.

EXAMPLE 7 Porous Particles with Carboxylic Groups

28.8 g of the seed latex of Example 1 was mixed with an emulsion of 6.4g dioctanoyl peroxide and 10.6 g acetone in 106.5 g 0.5% SDS in water.After stirring for 24 hours at room temperature, the reaction mixturewas added to an emulsion made from 20.7 g methacrylic acid, 83.0 gethylene glycol dimethacrylate, 103.7 g butyl acetate, 0.23 g SDS and975 g of a 0.8% solution of polyvinyl pyrrolidone in water. Thesuspension was stirred for 2 hours at room temperature and 470 g waterwas added. The temperature was raised slowly to 70° C. and the particleswere polymerised for 5 hours. After purification with methanol,monosized particles with diameters of 8.3 micrometers were obtained. Bynitrogen adsorption, the specific surface area was measured to be 210m²/g DS.

EXAMPLE 8 Superparamagnetic Particles from Carboxylic Particles

2.2 g FeCl₂×4H₂O and 3.0 g FeCl₃×6H₂O were dissolved in 106 g water andadded to a dispersion of 5 g dry substance of the particles of Example 7in 44 g water. The suspension was stirred for some minutes before 51 mlof 25% NH₃ in water was added. The temperature was raised to 80° C. for1 hour. The suspension was cooled and the particles were purified withwater by several cycles of centrifugation. After purification, theparticles were transferred to methanol and analysed by fluorescencemicroscopy. No autofluorescence could be observed under the sameconditions as would show considerable autofluorescence for conventionalmagnetic polymer particles. The particles were attracted to a magnet andthe content of iron was determined to 42 mg/g DS.

EXAMPLE 9 Porous Epoxy Particles

32.0 g of the particles of Example 1 was mixed with an emulsion of 6.8 gdioctanoyl peroxide and 11.3 g acetone in 114.1 g 0.5% SDS in water.After stirring for 24 hours at room temperature, the reaction mixturewas added to an emulsion of 57.6 g glycidyl methacrylate, 57.6 gtrimethylolpropane trimethacrylate, 115.2 g butyl acetate, 0.26 g SDSand 1085 g of a 1.1% solution of polyvinyl pyrrolidone in water. Thesuspension was stirred for 2 hours at room temperature and 521 g waterwas added. The temperature was raised slowly to 70° C. and the particleswere polymerised for 5 hours. After purification with methanol,monosized particles with diameters of 7.5 micrometers were obtained. Bynitrogen adsorption, the specific surface area was measured to 64 m2/gDS. Auto-fluorescence of these particles could not be observed byfluorescence microscopy.

EXAMPLE 10 Porous Amino Particles

78 g dry substance of the particles of Example 9, were transferred tomethanol and mixed with 1550 ml of 25% NH₃ in water. The suspension wasstirred at 60° C. overnight, before the particles were purified withwater. The content of primary amino groups was determined to be 0.8mmol/g DS by a ninhydrin reaction method.

EXAMPLE 11 Superparamagnetic Particles from Amino Particles

3.9 g FeCl₂×4H₂O and 7.9 g FeCl₃×6H₂O were dissolved in 100 g water andadded to a dispersion of 10 g dry substance of the particles of Example10 in 60 g water. The suspension was stirred for some minutes before 81ml of 25% NH₃ in water was added. The temperature was raised to 80° C.for 2 hours. The suspension was cooled and the particles were purifiedwith water by several cycles of centrifugation. After purification, theparticles were transferred to methanol and analysed by fluorescencemicroscopy. No autofluorescence could be observed under the sameconditions as would show considerable autofluorescence for conventionalmagnetic polymer particles. The particles were attracted to a magnet andshowed superparamagnetic behaviour. The content of iron was determinedto be 73 mg/g DS.

EXAMPLE 12 Measurement of Autofluorescence by Fluorescence MicroscopyInstrumentation and Software

The autofluorescence of superparamagnetic particles was measured with aBX-61 fluorescence microscope from Olympus. The microscope was equippedwith a 100 W mercury lamp and filter cubes from Chroma Technology. Theautofluorescence was measured through a filter cube with an excitationband pass-filter at 450 nm+/−25 nm. a beam splitter at 480 nm, andemission band pass filter at 520 nm. The objective used was an Uplan-Apo40×. The signal was detected with a F-view digital monochrome CCD camera(4096 grey levels). The exposure times were 600 ms in all the analyses.

The imaging analyses were performed with a software package, Analysis,from Soft Imaging Systems. The mean grey value of a particle population(MGV) is calculated as the mean of the mean intensity for each particle,resulting in an intensity value, that adjust for the differences inparticle area. The results are given as Mean Grey Value for theparticles. To obtain comparable values for the auto-fluorescence of thedifferent types of particles, the greyness values for the backgroundhave to be subtracted. The particle autofluorescence values aretherefore calculated as the difference in Mean Grey Values between theparticles and the background.

Sample Preparation

The particle samples were diluted with a 1:1 mixture by volume of 1.5 gg/L SDS in water and glycerol.

Two strips of a two-sided tape (3M Scotch 136 D) were attached to amicroscope slide, with a distance between the strips of approximately 3mm. A microscope cover glass was attached on the top of the strips togive a capillary channel between the slide and the cover glass. Thediluted particle samples were mounted into the cannels by capillaryforces, and the particles were allowed to settle before measurementswere performed.

Results

Two different types of uncoated magnetic polymer particles having astyrene/divinylbenzene matrix polymer and the uncoated particles ofExamples 3, 6, 8 and 11 were investigated. The results are given inTable 1.

In Table 2 the results are given for coated magnetic polymer particles.Three different types of coated magnetic polymer particles having astyrene/divinylbenzene matrix polymer and the coated particles ofExample 4 were investigated.

The autofluorescence of the particles is given as the difference (Δ MGV)between the Mean Grey Value of the particles and the Mean Grey Value ofthe background.

TABLE 1 Particle Auto- Raw data from fluorescence measurement given asMGV MGV Sample Description Δ MGV Particles Background A S/DVB 411 1016605 B S/DVB 411 1053 642 C Ex 3 83 678 595 D Ex 6 79 686 607 E Ex 8 51796 745 F Ex 11 62 595 533

TABLE 2 Particle Auto- Raw data from fluorescence measurement given asMGV MGV Sample Description Δ MGV Particles Background G S/DVB 769 1409640 H S/DVB 1423 2087 664 I S/DVB 1277 1905 628 J Example 4 287 971 684

EXAMPLE 13 Coating of Superparamagnetic Particles

0.5 gram of particles with diameter 5.1 μm, obtained by the methoddescribed in example 11, were transferred to 9.5 gram diglyme. 4.6 gramglycidol was added. The suspension was shaken for 20 hours at 75° C. Theparticles were purified with diglyme and methanol. FT-IR adsorption of1080 cm⁻¹ confirmed appearance of ether coating from the epoxide. Theparticles were analysed by fluorescence microscopy.

EXAMPLE 14 Coating of Superparamagnetic Particles

0.5 gram of particles with diameter 5.1 μm, obtained by the methoddescribed in example 11, were transferred to 9.5 gram toluene. 3.6 grambutandiol diglycidylether and 8.3 ethyl hexyl glycidylether were added.The suspension was shaken for 20 hours at 75° C. The particles werepurified with toluene and methanol. FT-IR adsorption of 1080 cm⁻¹confirmed appearance of ether coating from the epoxides. The particleswere analysed by fluorescence microscopy.

EXAMPLE 15 Coating of Superparamagnetic Particles

0.5 gram of particles with diameter 5.1 μm, obtained by the methoddescribed in example 11, were transferred to 9.5 gram diglyme. 2.3 grambutyl glycidylether and 3.3 gram glycidol were added. The suspension wasshaken for 20 hours at 75° C. The particles were purified with diglymeand methanol. FT-IR adsorption of 1080 cm⁻¹ confirmed appearance ofether coating from the epoxides. The particles were analysed byfluorescence microscopy.

EXAMPLE 16 Coating of Superparamagnetic Particles

0.5 gram of particles with diameter 5.1 μm, obtained by the methoddescribed in example 11, were transferred to 9.5 gram toluene. 3.3 gglycidol and 3.6 g butandiol diglycidylether were added. The suspensionwas shaken for 20 hours at 75° C. The particles were purified withtoluene and methanol. FT-IR adsorption of 1080 cm⁻¹ confirmed appearanceof ether coating from the epoxides. The particles were analysed byfluorescence microscopy.

EXAMPLE 17 Amino Particles

15 gram particles, obtained by the methods described in example 16, wasadded to a mixture of 135 gram diglyme and 135 gram ethylenedioxydiethylamine. The dispersion was heated to 60° C. After 4 hours theparticles were purified with methanol and anhydrous dimethylformamide.Primary amino groups in the particles were verified by a ninhydrinreaction method. The particles were analysed by fluorescence microscopyand flowcytometry.

EXAMPLE 18 Measurement of Autofluorescence by Fluorescence MicroscopyInstrumentation and Software

The autofluorescence of superparamagnetic particles from Examples 13-17was measured with a BX-61 fluorescence microscope from Olympus. Themicroscope was equipped with a 75 W xenon lamp and filter cubes fromChroma Technology. The autofluorescence was measured through threedifferent filter cubes using the exposure times as shown in table 3. Theobjective used was an Uplan-Apo 20×. The signal was detected with anF-view digital monochrome CCD camera (4096 grey levels). The imaginganalyses were performed with a software package, Analysis, from SoftImaging Systems. The mean grey value of a particle population (MGV) iscalculated as the mean of the mean intensity for each particle resultingin an intensity value, which adjusts for the differences in particlearea. The results are given as Mean Grey Value for the particles. Toobtain comparable values for the autofluorescence of the different typesof particles, the greyness values for the background have to besubtracted. The particle autofluorescence values are thereforecalculated as the difference in Mean Grey Values between the particlesand the background.

TABLE 3 Filter Settings Exposure Filter Beam Time Cube ExcitationSplitter Emission  500 ms 1 450 (50) nm 480 nm 520 nm long pass  5000 ms2 550 (30) nm 580 nm 620 (60) nm 50000 ms 3 630 (30) nm 650 nm 665 nmlong pass

Sample Preparation

All the particles were diluted with phosphate buffer saline (pH 7.4)before transferring to a capillary channel of the microscope slide, asdescribed in example 12.

Results

Two different types of coated magnetic polymer particles having astyrene/divinylbenzene matrix polymer and the coated particles ofExamples 13, 14, 15, 16 and 17 were investigated. The results given inTable 4 are also compared to commercial available calibration particles,Certified blanks.

The Certified blank particles are included in MESF calibration setobtained from Bangs Laboratories, Inc. The particles are 7.4 micrometercompact non-magnetic polymer particles, which may be used asnon-fluorescent references for flowcytometry applications as describedby i.e. Schwartz in U.S. Pat. No. 5,089,416.

TABLE 4 Δ MGV Δ MGV Δ MGV Filter Filter Filter Sample Description Cube 1Cube 2 Cube 3 A Blank −1 −1 −67 B PS/DVB 1087 1851 1269 C PS/DVB 833 829426 D EX 13 333 31 −72 E EX 14 77 18 7 F Ex 15 11 14 −51 G Ex 16 1 67−64 H Ex 17 53 35 −45

EXAMPLE 19 Measurement of Autofluorescence by FlowcytometryInstrumentation and Software

The autofluorescence of superparamagnetic particles from Example 17 wasmeasured with BD LSR II flowcytometer from BD biosciences. Theexcitation source was a 488 nm solid-state laser and a 633 nm heliumneon laser. The excitation and filter specifications for the differentfluorescence detectors are summarized in table 5. The voltage of the PMTdetectors was all set to 600 V. The statistics were carried out bysetting a gate in the plot of forward scatter (FSC) against side scatter(SSC), which included the single particles only. The flowcytometer wasset to count 10 000 particles with an approximate rate of 100 events persecond for each sample. The area-mean was determined for each sample.For area detection the signal will decrease with a decrease in size forthe same kind of particle. (Because the intensity from the flowmeasurement is an integral of the detected intensity profile generatedwhen the particle is passing the objective.)

TABLE 5 Filter Settings of BD LSR II flow cytometer Excitation EmissionSource Beam Splitter Filter Detector 488 nm 505 nm long pass 530 (30) nm1 laser 550 nm long pass 575 (26) nm 2 685 nm long pass 695 (40) nm 3735 nm long pass 780 (60) nm 4 633 nm blank 660 (20) nm 5 laser 735 nmlong pass 780 (60) nm 6

Sample Preparation

All samples were diluted with phosphate buffer saline (PBS pH 7.4) priorto the analysis. The particle concentration was adjusted to give anapproximate count of 100 events per second by the flowcytometer.

Results

Two different types of coated magnetic polymer particles havingstyrene/divinylbenzene and the coated particles of example 17 wereinvestigated. The results given in Table 6 are also compared tocommercial available calibration particles, Certified blanks.

The Certified blank particles are included in MESF calibration setobtained from Bangs Laboratories, Inc. The particles are 7.4 micrometercompact non-magnetic polymer particles, which may be used asnon-fluorescent references for flowcytometry applications as describedby i.e. Schwartz in U.S. Pat. No. 5,089,416.

TABLE 6 Intensity: mean channel number of singlets Flow Measured bydetector Cytometry #1 #2 #3 #4 #5 #6 Blank 246 32 14 2 100 3 PS/DVB 3040784 1088 416 212 50 PS/DVB 2166 492 471 149 87 10 Ex 17 579 84 26 4 31 0

By use of MESF calibration kits for fluorescein isothiocyanate (FITC),phycoerythrin (PE) and PE-Cy5, received from Bangs Laboratories Inc, themeasured autofluorescence can be assigned to values of Molecules ofEquivalent Soluble Fluorophore (MESF). The MESF definition may be foundat http://nvl.nist.gov/pub/nistpubs/jres/107/1/j71schw.pdf

In Table 7 the estimated MESF values when comparing the autofluorescenceto the respective calibration curves are given.

TABLE 7 Excitation Source 488 nm Laser Emission filter 530 (30) 575 (26)695 (40) MESF MESF MESF FITC PE PE-Cy5 Ex 17 1342 362 66 PS/DVB 72253625 2635 PS/DVB 5122 2242 1151 Blank 563 134 36

EXAMPLE 20 Incorporation of Bodipy 630/650 Giving SuperparamagneticParticles with Three Levels of Fluorescence Intensity

A 10% (w/w) dispersion in dimethyl formamide of 8 μm particles, obtainedby the method described in example 17, was transferred to three vials A,B and C. 5.6 mg Bodipy 630/650 succinimidyl ester (from Molecular ProbesInc.) was mixed with 11.9 mL dimethylformamide and added to the particledispersions in the vials. In vial A, 50 g particle dispersion was mixedwith 10.6 mL colour solution, in vial B 30 g particle dispersion wasmixed with 0.6 mL colour solution and in vial C 30 g particlesdispersion was mixed with 0.06 mL colour solution. The mixtures wereshaken for 24 hours at ambient temperature and then placed on a magnet.The particles were purified with methanol, 5% triethylamine in methanoland water and diluted in phosphate buffer saline (PBS, pH 7.4). Theparticles were analyzed by fluorescence microscopy and flowcytometry.

Measurement by Fluorescence Microscopy

The microscopy measurements were performed with a BX-61 fluorescencemicroscope from Olympus, equipped with a 75 W Xenon lamp and filtercubes from Chroma Technology. The fluorescence was measured through afilter cube with excitation bandpass filter at 630(30) nm, beamsplitterat 650 nm, and emission longpass filter at 665 nm. The objective usedwas Uplan-Apo 20×. The camera exposure time settings were adjusted tothe corresponding amount of dye attached to the particles.

All the particles were diluted with phosphate buffer saline (pH 7.4) andtransferred to a capillary channel of the microscope slide, as describedin example 12.

The resulting fluorescence intensities given as AMGV were calculated asin example 12. The resulting coefficient of variation (% CV) is definedas the standard deviation of mean grey value (MGV) of particles dividedby the mean grey value (MGV) of particles multiplied by a factor of 100.The coloured particles were compared with similar particles without dyeincorporated (blank references).

TABLE 8 Exposure MGV MGV Microscopy time (ms) Particle Background ΔMGVCV % Ex 20, A 50 2349 76 2273 10 Ex 20, B 50 425 64 361 10 Ex 20, C 100153 64 89  8 Blank 1000 85 96 −11 —

Measurements by Flowcytometry

The flowcytometry measurements were performed with a BD LSR IIflowcytometer from Becton-Dickinson. The instrument was equipped with a633 nm laser as excitation source. For detection an emission bandpassfilter with specification 660(20) nm, corresponding to the APC(Allophycoerithrin) filter-settings for the BD LSR II flowcytometer, wasused. The voltage of the PMT detector was set to 250 V. The statisticswere carried out by setting a gate in the plot of forward scatter (FSC)against side scatter (SSC), including single particles only. Theflowcytometer was set to count 10 000 particles with an approximate rateof 100 events per second for each sample. The single particle populationcontained typically 90% of the total number of events. The area mean andcoefficient of variation was determined for each sample. The coefficientof variation (% CV) is defined as the standard deviation of singletsdivided by the mean intensity of singlets, at that specific voltage,multiplied by a factor of 100.

The coloured particles were compared with similar particles without dyeincorporated, named blank references. All samples were diluted withphosphate buffer saline (PBS pH 7,4) prior to the analysis. The particleconcentration was adjusted to give an approximate count of 100 eventsper second by the flowcytometer. The results are given in Table 9.

TABLE 9 Intensity: Mean channel no. Intensity Flow singlets PMTDistribution Cytometry Voltage = 250 v CV % Ex 20, A 8848 16 Ex 20, B1536 16 Ex 20, C 153 42 Blank 0 —

EXAMPLE 21 Incorporation of Bodipy 650/665 Giving SuperparamagneticParticles with Three Levels of Fluorescence Intensity

5 μm particles obtained with the method described in example 17 weretransferred into dimethylformamide giving 10% w/w dispersion. 1.5 mL ofthe dispersion was transferred to three vials each, A, B and C. 1.1 mgBodipy 650/665 succinimidyl ester (from Molecular Probes Inc.) was mixedwith 1.25 mL dimethylformamide. The Bodipy solution was mixed with theparticles dispersions giving the following amount of dye per mg drysubstance (DS) of particles; in A) 1.0 mg/g DS, in B) 0.1 mg/g DS and inC) 0.01 mg/g DS. The dispersions were shaken at ambient temperature overnight. The particles were purified with a solution of 5% triethylaminein methanol and phosphate buffer saline (PBS, pH 7.4).

The particles were analyzed by fluorescence microscopy and flowcytometryas described in Example 20. The results given in Table 10 and Table 11demonstrate that particle populations can be coloured with a largedifference in intensity also for this type of dye.

TABLE 10 Exposure MGV MGV Microscopy time (ms) Particle Background ΔMGVCV % Ex 21, A 10 511 64 447 8 Ex 21, B 100 758 76 682 10  Ex 21, C 1000826 96 730 9 Blank 50000 1113 1148 −35 —

TABLE 11 Intensity: Mean channel no. Intensity Flow singlets PMTDistribution Cytometry Voltage = 250 v CV % Ex 21, A 1133 15 Ex 21, B192 15 Ex 21, C 20 73 Blank 0 —

EXAMPLE 22 Incorporation of DY-647 Giving Superparamagnetic Particleswith Three Levels of Fluorescence Intensity

5 μm particles obtained with the method described in example 17 wastransferred into dimethylformamide giving 10% w/w dispersion. 1.5 mL ofthe dispersion was transferred to three vials each, A, B and C. 1.1 mgDY-647 succinimidyl ester (from Dyomics GmbH) was mixed with 1.25 mLdimethylformamide. The dye solution was mixed with the particlesdispersions giving the following amount of dye per mg dry substance (DS)of particles; in A) 1.0 mg/g DS, in B) 0.1 mg/g DS and in C) 0.01 mg/gDS. The dispersions were shaken at ambient temperature over night. Theparticles were purified with a solution of 5% triethylamine in methanol,water and diluted in phosphate buffer saline (PBS, pH 7.4).

The particles were analyzed by fluorescence microscopy and flowcytometryas described in Example 20. The results, given in Table 12 and Table 13,demonstrate that particle populations can be coloured with a largedifferences in intensity also for this type of dye (DY-647).

TABLE 12 Exposure MGV MGV Microscopy time (ms) Particle Background ΔMGVCV % Ex 22, A 10 1135 65 1070 15 Ex 22, B 100 1180 81 1099  8 Ex 22, C1000 1298 98 1200 10 Blank 50000 1113 1148 −35 —

TABLE 13 Intensity: Mean channel no. Intensity Flow singlets PMTDistribution Cytometry Voltage = 250 v CV % Ex 22, A 2955 14 Ex 22, B356 9 Ex 22, C 38 227 Blank 0 —

EXAMPLE 23 Fluorescent Particles with Narrow Intensity Distribution

5 μm particles obtained by the method described in example 17 weretransferred to dimethylformamide. 15 gram of 10% (w/w) particledispersion was mixed with 1.2 mg of DY-647 succinimidylester dissolvedin 2 mL dimethylformamide. After 2 hours a small sample of the colouredparticles was purified with methanol and phosphate buffer saline (PBS,pH 7.4). The sample were analysed on the flowcytometer as described inexamples 20, 21 and 22 with one exception, use of slower sheath fluidflow (low setting). The result, given in Table 14, shows that thesuperparamagnetic beads can be coloured to give particle populationswith very narrow intensity distribution.

TABLE 14 Intensity: Mean channel no. Intensity Flow singlets PMTDistribution Cytometry Voltage = 250 v CV % A 2336 6

EXAMPLE 24 Colouring Prior to Coating Colouring Procedure

Particles obtained by the method described in Example 11 weretransferred to anhydrous dimethylformamide. In four different bottles,38 gram of 5% particle dispersion was mixed with 4 mL of dimethylformamide containing 2 mg dye. The four fluorescent dyes used, AMCA-X,Bodipy TMR, Bodipy FL (from Molecular Probes Inc) and DY?555 (fromDyomics GmbH) had succinimidyl ester functionality. After stirring for20 hours at ambient temperature, the particles were purified severaltimes with 5% triethylamine in methanol and finally with methanol. Priorto microscopy analyses, small samples of particles were diluted inphosphate buffer saline (pH 7.4).

Table 15 shows the fluorescence intensities of dyed particles as theDMGV value, analyzed in appropriate filter for the specific fluorophoreattached to the particles.

Coating Procedure

0.3 gram of the coloured particles above, were transferred to diethyleneglycol dimethyl ether. The content of particles in the dispersions werejustified to 10% dry substance (DS). 2.1 gram 1,4-Butandioldiglycidylether and 2.2 gram glycidol were added to the particlesuspensions. The mixtures were shaken on vortex and placed in a shaker(LabMate) at 75° C. for 20 hours. The particles were purified twice withmethanol and with phosphate buffer saline (pH 7.4). Prior to microscopyanalyses small samples of particles were diluted with phosphate buffersaline (pH 7.4).

Table 15 shows the fluorescence intensities of dyed and coated particlesas the DMGV value, analyzed in appropriate filter for the specificfluorophore attached to the particles. The results indicate that thecoumarin-dye, AMCA-X (from Molecular Probes Inc) may be a betteralternative than a series of other dyes when the coating step is doneafter the colouring.

TABLE 15 ΔMGV ΔMGV (Dyed and (Dyed coated Excitation Emission ExposureDye particle) particle) filter nm filter nm time AMCA-X 470 614 360 (40)420 LP 10 Bodipy 1137 66 550 (30) 620 (60) 10 TMR Bodipy 1710 174 450(50) 520 LP 10 FL DY-555 2492 40 550 (30) 620 (60) 20

By microscopy, particles coloured with AMCA-X and coated were comparedto equivalent particles without dye, blank references, detected throughdifferent filters. As can be seen in Table 16, the AMCA-dye does notdisturb the detection in filters suitable for a reporter signals

TABLE 16 Excitation 360 (40) 450 (50) 550 (30) 630 (30) filter (nm)Emission 420 LP 520 LP 620 (60) 665 LP filter (nm) Exposure time 10 ms500 ms 5000 ms 50000 ms Particle type ΔMGV ΔMGV ΔMGV ΔMGV Blankreference  0  90 51 −186 Dyed with AMCA 584 108 17 −110 and coated

1. A coated magnetic particle comprising an optionally porous magneticpolymer particle of a matrix polymer, said polymer particle having on asurface and/or in the pores thereof superparamagrietic crystals, saidcoated particle having a coefficient of variation (CV) of less than 10%and having a coat formed of a coating polymer, wherein said coatedmagnetic particle is essentially non-autofluorescent.
 2. The coatedmagnetic particle as claimed in claim 1 having a level ofautofluorescence corresponding to a difference in the Mean Grey Value(ΔMGV) at an excitation wavelength of 450±25 nm for an exposure time of500 ms or 600 ms of less than
 600. 3. The coated magnetic particle asclaimed in claim 2 having a level of autofluorescence corresponding to adifference in the Mean Grey Value (ΔMGV) at an excitation wavelength of450±25 nm for an exposure time of 500 ms or 600 ms of less than
 300. 4.The coated magnetic particle as claimed in claim 1 being essentiallyfree of conjugated delocalized electron systems (except those withinbenzene rings).
 5. The coated magnetic particle as claimed in claim 1wherein said coating polymer is formed from at least one epoxide.
 6. Thecoated magnetic particle as claimed in claim 5 wherein said coatingpolymer is formed from at least two epoxides.
 7. The coated magneticparticle as claimed in claim 6 wherein the epoxides are selected fromepichlorohydrin, epibromohydrin, isopropylglycidyl ether, butyl glycidylether, allylglycidyl ether, 1,4-butanediol diglycidyl ether(1,4-bis(2,3-epoxypropoxy)butane), ethylhexylglycidylether, methylglycidylether, neopentylglycol diglycidyl ether, ethylene glycoldiglycidyl ether, glycerol diglycidyl ether, glycidol, and glycidylmethacrylate.
 8. The coated magnetic particle as claimed in claim 1wherein the polymer particle is porous.
 9. The coated magnetic particleas claimed in claim 1 wherein the polymer particle has a diameter in therange 0.8 to 10 μm.
 10. The coated magnetic particle as claimed in claim9 wherein the particle is in the range 2 to 10 μm in diameter.
 11. Thecoated magnetic particle as claimed in claim 1 wherein said matrixpolymer is a combination of a styrenic polymer, a styrenic/acrylicpolymer and an acrylic polymer.
 12. The coated magnetic particle asclaimed in claim 1 wherein the particle surface carries free aminogroups.
 13. The coated magnetic particle as claimed in claim 1additionally comprising a fluorophore or mixture of differentfluorophores.
 14. The coated magnetic particle as claimed in claim 13wherein said fluorophore is attached to the external coating polymer viaan amino linkage or is swollen into the coating polymer and entrappedtherein.
 15. The coated magnetic particle as claimed in claim 13 whereinsaid fluorophore is Bodipy 630/650, Bodipy 650/655, DY-647 or AMCA-X.16. The coated magnetic particle as claimed in claim 13 wherein saidfluorophore emits in the blue/green region of the electromagneticspectrum.
 17. The coated magnetic particle as claimed in claim 1 orclaim 13 additionally comprising an affinity ligand.
 18. A mixture ofcoated magnetic particles as claimed in claim 1 wherein the CV of thepolymer particle diameters is less than 8%.
 19. The mixture of coatedmagnetic particles as claimed in claim 18 comprising at least a firstset of coated magnetic particles carrying a first fluorophore and secondset of coated magnetic particles carrying a second fluorophore, theparticles, of said first and second sets being of differing diameters.20. A process for the preparation of coated magnetic particles which arefree of conjugated delocalised electron systems, comprising the stepsof: (a) providing a solution comprising porous magnetic polymerparticles which are essentially free from surface nitro groups andhaving superparamagnetic crystals on the surface and/or in the poresthereof, (b) providing a solution comprising polymer-forming monomersselected from epoxides, (c) combining the solution comprising themagnetic polymer particles with the solution comprising apolymer-forming monomer, and (d) isolating the coated magneticparticles.
 21. (canceled)
 22. An assay method for detecting the presenceand/or amount of an analyte in a sample wherein said assay comprise thesteps of: i) adding a coated magnetic particle as claimed in claim 1 andfurther comprising a first fluorophore and an affinity ligand to asample in which the target analyte may be present; ii) allowing saidaffinity ligand to bind with said analyte if present to form a complex;iii) contacting the complex with a reporter to allow binding of saidreporter to said analyte wherein said reporter optionally carries asecond fluorophore; iv) magnetically separating said particles from saidsample; and v) detecting said first fluorophore and if present saidsecond fluorophore.
 23. The method as claimed in claim 22 whereindetection is carried out using a flow cytometer or by microscopy. 24.The method as claimed in claim 22 wherein the particles form part of abead array, which is analysed to detect the position of each bead typeand the presence of second fluorophore.
 25. The method as claimed inclaim 22 wherein said reporter is biotin or a second fluorophore whichis different from said first fluorophore.
 26. The method as claimed inclaim 22 wherein step (i) additionally comprises the step of: adding atleast a second coated magnetic particle carrying a further affinityligand (specific for other analytes which may be present in the sample)and a further fluorophore to said sample, said first and furtheraffinity ligands and said first and further fluorophores beingdifferent.
 27. The method as claimed in claim 26 wherein said secondcoated magnetic particle is of different diameter to said first coatedmagnetic particle.
 28. The method as claimed in claim 22 wherein saidreporter is biotin and said complexes are contacted with a streptavidincoated surface prior to fluorophore detection.
 29. An assay method fordetecting the presence and/or amount of an analyte in a sample whereinsaid assay comprises the steps of: i) contacting a coated magneticparticle comprising a first fluorophore and an affinity ligand with thesample in which a target analyte may be present and in which an analytecarrying a reporter capable of competing with said target analyte forthe affinity ligand is present; ii) allowing said affinity ligand tobind to said analyte, if present, or said analyte carrying a reporter;iii) magnetically separating said particles from said sample; and iv)detecting the first fluorophore.
 30. The method as claimed in claim 29wherein said reporter is biotin and said complexes are contacted with astreptavidin coated surface prior to fluorophore detection.
 31. Anoptionally porous magnetic polymer particle comprising a matrix polymer,with disposed on the surface and/or in the pores thereofsuperparamagnetic crystals, said matrix polymer being essentially freeof conjugated delocalized electron systems and optionally being surfacefunctionalised with groups selected from sulphonic acid, carboxylicacid, amine, and epoxy groups, said particle being essentiallynon-autofluorescent.